卵形鲳鲹JAK3蛋白的原核表达与纯化
S913; [目的]纯化获取卵形鲳鲹(Trachinotus ovatus)JAK3(TroJAK3)重组蛋白,为了解TroJAK3蛋白功能、相互作用及抗体制备奠定基础.[方法]应用分子生物学技术构建重组质粒pET-32a-TroJAK3,转化大肠杆菌BL21感受态细胞,经IPTG诱导表达,SDS-PAGE和Western blot检测TroJAK3重组蛋白表达情况.[结果]PCR扩增片段长度为3 333 bp,经双酶切鉴定、测序确认序列和开放阅读框正确,结果显示成功构建重组质粒pET-32a-TroJAK3.经终浓度为1 mmol/L IPTG 37℃诱导4 h后进行SDS-PAGE检测,结...
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| Published in | 广东农业科学 Vol. 47; no. 1; pp. 137 - 142 |
|---|---|
| Main Authors | , , , , , |
| Format | Journal Article |
| Language | Chinese |
| Published |
广西大学动物科学技术学院,广西 南宁,530004%广西水产科学研究院/广西水产遗传育种与健康养殖重点实验室,广西 南宁,530021
2020
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| Subjects | |
| Online Access | Get full text |
| ISSN | 1004-874X |
| DOI | 10.16768/j.issn.1004-874X.2020.01.019 |
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| Abstract | S913; [目的]纯化获取卵形鲳鲹(Trachinotus ovatus)JAK3(TroJAK3)重组蛋白,为了解TroJAK3蛋白功能、相互作用及抗体制备奠定基础.[方法]应用分子生物学技术构建重组质粒pET-32a-TroJAK3,转化大肠杆菌BL21感受态细胞,经IPTG诱导表达,SDS-PAGE和Western blot检测TroJAK3重组蛋白表达情况.[结果]PCR扩增片段长度为3 333 bp,经双酶切鉴定、测序确认序列和开放阅读框正确,结果显示成功构建重组质粒pET-32a-TroJAK3.经终浓度为1 mmol/L IPTG 37℃诱导4 h后进行SDS-PAGE检测,结果表明TroJAK3重组蛋白以包涵体形式大量表达,分子量约为140 ku.经Ni-IDA树脂柱纯化,获得纯化的重组蛋白.Western blot检测结果显示有140 ku的条带,表明TroJAK3重组蛋白能被抗His抗体识别.[结论]成功构建了重组质粒pET-32a-TroJAK3,纯化得到高纯度的TroJAK3融合蛋白. |
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| AbstractList | S913; [目的]纯化获取卵形鲳鲹(Trachinotus ovatus)JAK3(TroJAK3)重组蛋白,为了解TroJAK3蛋白功能、相互作用及抗体制备奠定基础.[方法]应用分子生物学技术构建重组质粒pET-32a-TroJAK3,转化大肠杆菌BL21感受态细胞,经IPTG诱导表达,SDS-PAGE和Western blot检测TroJAK3重组蛋白表达情况.[结果]PCR扩增片段长度为3 333 bp,经双酶切鉴定、测序确认序列和开放阅读框正确,结果显示成功构建重组质粒pET-32a-TroJAK3.经终浓度为1 mmol/L IPTG 37℃诱导4 h后进行SDS-PAGE检测,结果表明TroJAK3重组蛋白以包涵体形式大量表达,分子量约为140 ku.经Ni-IDA树脂柱纯化,获得纯化的重组蛋白.Western blot检测结果显示有140 ku的条带,表明TroJAK3重组蛋白能被抗His抗体识别.[结论]成功构建了重组质粒pET-32a-TroJAK3,纯化得到高纯度的TroJAK3融合蛋白. |
| Author | 杨萌 段家文 谢业扬 高爽爽 陆专灵 韦友传 |
| AuthorAffiliation | 广西大学动物科学技术学院,广西 南宁,530004%广西水产科学研究院/广西水产遗传育种与健康养殖重点实验室,广西 南宁,530021 |
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| Author_FL | LU Zhuanling DUAN Jiawen GAO Shuangshuang WEI Youchuan XIE Yeyang YANG Meng |
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| DocumentTitle_FL | Prokaryotic Expression and Purification of JAK3 Protein in Golden Pompano(Trachinotus ovatus) |
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| Keywords | 卵形鲳鲹 蛋白纯化 原核表达 JAK3蛋白 |
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| Title | 卵形鲳鲹JAK3蛋白的原核表达与纯化 |
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